Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides

ABSTRACT

The invention relates to the therapeutic use of oligonucleotides as immunostimulatory agents in immunotherapy applications. More particularly, the invention provides an immunostimulatory oligonucleotides for use in methods for generating an immune response or for treating a patient in need of immunostimulation. The immunostimulatory oligonucleotides of the invention preferably comprise novel purines. The immunostimulatory oligonucleotides according to the invention further comprise at least two oligonucleotides linked at their 3′ ends, internucleoside linkages or functionalized nucleobase or sugar to a non-nucleotidic linker, at least one of the oligonucleotides being an immunostimulatory oligonucleotide and having an accessible 5′ end.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to immunology and immunotherapy applications usingoligonucleotides as immunostimulatory agents.

2. Summary of the Related Art

Oligonucleotides have become indispensable tools in modern molecularbiology, being used in a wide variety of techniques, ranging fromdiagnostic probing methods to PCR to antisense inhibition of geneexpression and immunotherapy applications. This widespread use ofoligonucleotides has led to an increasing demand for rapid, inexpensiveand efficient methods for synthesizing oligonucleotides.

The synthesis of oligonucleotides for antisense and diagnosticapplications can now be routinely accomplished. See, e.g., Methods inMolecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogspp. 165-189 (S. Agrawal, ed., Humana Press, 1993); Oligonucleotides andAnalogues, A Practical Approach, pp. 87-108 (F. Eckstein, ed., 1991);and Uhlmann and Peyman, supra; Agrawal and Iyer, Curr. Op. in Biotech.6:12 (1995); and Antisense Research and Applications (Crooke and Lebleu,eds., CRC Press, Boca Raton, 1993). Early synthetic approaches includedphosphodiester and phosphotriester chemistries. For example, Khorana etal., J. Molec. Biol. 72:209 (1972) discloses phosphodiester chemistryfor oligonucleotide synthesis. Reese, Tetrahedron Lett. 34:3143-3179(1978), discloses phosphotriester chemistry for synthesis ofoligonucleotides and polynucleotides. These early approaches havelargely given way to the more efficient phosphoramidite andH-phosphonate approaches to synthesis. For example, Beaucage andCaruthers, Tetrahedron Lett. 22:1859-1862 (1981), discloses the use ofdeoxyribonucleoside phosphoramidites in polynucleotide synthesis.Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), disclosesoptimized synthesis of oligonucleotides by the H-phosphonate approach.Both of these modern approaches have been used to synthesizeoligonucleotides having a variety of modified internucleotide linkages.Agrawal and Goodchild, Tetrahedron Lett. 28:3539-3542 (1987), teachessynthesis of oligonucleotide methylphosphonates using phosphoramiditechemistry. Connolly et al., Biochem. 23:3443 (1984), discloses synthesisof oligonucleotide phosphorothioates using phosphoramidite chemistry.Jager et al., Biochem. 27:7237 (1988), discloses synthesis ofoligonucleotide phosphoramidates using phosphoramidite chemistry.Agrawal et al., Proc. Natl. Acad. Sci. (USA) 85:7079-7083 (1988),discloses synthesis of oligonucleotide phosphoramidates andphosphorothioates using H-phosphonate chemistry.

More recently, several researchers have demonstrated the validity of theuse of oligonucleotides as immunostimulatory agents in immunotherapyapplications. The observation that phosphodiester and phosphorothioateoligonucleotides can induce immune stimulation has created interest indeveloping this side effect as a therapeutic tool. These efforts havefocused on phosphorothioate oligonucleotides containing the dinucleotidenatural CpG. Kuramoto et al., Jpn. J. Cancer Res. 83:1128-1131 (1992)teaches that phosphodiester oligonucleotides containing a palindromethat includes a CpG dinucleotide can induce interferon-alpha and gammasynthesis and enhance natural killer activity. Krieg et al., Nature371:546-549 (1995) discloses that phosphorothioate CpG-containingoligonucleotides are immunostimulatory. Liang et al., J. Clin. Invest.98:1119-1129 (1996) discloses that such oligonucleotides activate humanB cells. Moldoveanu et al., Vaccine 16:1216-124 (1998) teaches thatCpG-containing phosphorothioate oligonucleotides enhance immune responseagainst influenza virus. McCluskie and Davis, J. Immunol. 161:4463-4466(1998) teaches that CpG-containing oligonucleotides act as potentadjuvants, enhancing immune response against hepatitis B surfaceantigen. Hartman et al., J. Immunol. 164: 1617-1624 (2000) teaches thatthe immunostimulatory sequence is species specific, and differentbetween mice and primates.

Other modifications of CpG-containing phosphorothioate oligonucleotidescan also affect their ability to act as modulators of immune response.See, e.g., Zhao et al., Biochem. Pharmacol. (1996) 51:173-182; Zhao etal., Biochem Pharmacol. (1996) 52:1537-1544; Zhao et al., AntisenseNucleic Acid Drug Dev. (1997) 7:495-502; Zhao et al., Bioorg. Med. Chem.Lett. (1999) 9:3453-3458; Zhao et al., Bioorg. Med. Chem. Lett. (2000)10:1051-1054; Yu et al., Bioorg. Med. Chem. Lett. (2000) 10:2585-2588;Yu et al., Bioorg. Med. Chem. Lett. (2001) 11:2263-2267; and Kandimallaet al., Bioorg. Med. Chem. (2001) 9:807-813.

These reports make clear that there remains a need to be able tomodulate the immune response caused by immunostimulatoryoligonucleotides and to overcome species specificity of theimmunostimulatory sequences.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods for modulating the immune response causedby oligonucleotide compounds. The methods according to the inventionenable modifying the cytokine profile produced by immunostimulatoryoligonucleotides for immunotherapy applications. The present inventorshave surprisingly discovered that modification of immunostimulatorydinucleotides allows flexibility in the nature of the immune responseproduced and that certain modifications overcome the speciesspecificities observed to date of the immunostimulatory sequences.

In a first aspect the invention provides an immunostimulatoryoligonucleotide having a structure from the group of5′-TCTGTR′GTTCT-X-TCTTGR′TGTCT-5′;5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′;5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′;5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′;5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′;5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′; 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′;5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′;5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′; 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′;5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′;5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′;5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′; 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′and 5′-TCRAACRTTCR-X-RCTTRCAARCT-5′, whereinR′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁=2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine andX=glycerol linker.

In a second aspect the invention provides pharmaceutical compositions.These compositions comprise any one of the compositions disclosed in thefirst aspect of the invention and a pharmaceutically acceptable carrier.

In a third aspect the invention provides a method for generating animmune response in a vertebrate, the method comprising administering tothe vertebrate an immunostimulatory oligonucleotide having a structurefrom the group of 5′-TCTGTR′GTTCT-X-TCTTGR′TGTCT-5′;5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′;5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′;5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′;5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′;5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′; 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′;5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′;5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′; 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′;5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′;5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′;5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′; 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′and 5′-TCRAACRTTCR-X-RCTTRCAARCT-5′, whereinR′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁=2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine andX=glycerol linker.

In a fourth aspect the invention provides a method for therapeuticallytreating a vertebrate having cancer, an autoimmune disorder, airwayinflammation, inflammatory disorders, skin disorders, allergy, asthma ora disease caused by a pathogen, such method comprising administering tothe patient an immunostimulatory oligonucleotide having a structure fromthe group of 5′-TCTGTR′GTTCT-X-TCTTGR′TGTCT-5′;5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′;5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′;5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′;5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′;5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′; 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′;5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′;5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′; 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′;5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′;5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′;5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′; 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′and 5′-TCRAACRTTCR-X-RCTTRCAARCT-5′, whereinR′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁=2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine andX=glycerol linker.

In a fifth aspect the invention provides a method for preventing cancer,an autoimmune disorder, airway inflammation, inflammatory disorders,skin disorders, allergy, asthma or a disease caused by a pathogen in avertebrate, such method comprising administering to the vertebrate animmunostimulatory oligonucleotide having a structure from the group of5′-TCTGTR′GTTCT-X-TCTTGR′TGTCT-5′;5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′;5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′;5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′;5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′;5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′; 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′;5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′;5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′; 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′;5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′;5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′;5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′; 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′and 5′-TCRAACRTTCR-X-RCTTRCAARCT-5′, whereinR′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁=2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine andX=glycerol linker.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a group of representative small molecule linkers suitablefor linear synthesis of immunostimulatory oligonucleotides of theinvention.

FIG. 2 depicts a group of representative small molecule linkers suitablefor parallel synthesis of immunostimulatory oligonucleotides of theinvention.

FIG. 3 is a synthetic scheme for the linear synthesis ofimmunostimulatory oligonucleotides of the invention.DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 4 is a synthetic scheme for the parallel synthesis ofimmunostimulatory oligonucleotides of the invention.DMTr=4,4′-dimethoxytrityl; CE=cyanoethyl.

FIG. 5 is a schematic representation of the 3′-terminal nucleoside of anoligonucleotide, showing that a non-nucleotidic linkage can be attachedto the nucleoside at the nucleobase, at the 3′ position, or at the 2′position.

FIG. 6 shows IL-12 induction in C57BL/6 mouse spleen cell cultures byimmunostimulatory oligonucleotides of the invention.

FIG. 7 shows IL-6 induction in C57BL/6 mouse spleen cell cultures byimmunostimulatory oligonucleotides of the invention.

FIG. 8 shows IFN-α induction in human pDC cultures by immunostimulatoryoligonucleotides of the invention.

FIG. 9 shows IFN-α induction in human PBMC cultures by immunostimulatoryoligonucleotides of the invention.

FIG. 10 shows Human B cell proliferation by immunostimulatoryoligonucleotides of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic use of oligonucleotides asimmunostimulatory agents for immunotherapy applications. The issuedpatents, patent applications, and references that are cited herein arehereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.In the event of inconsistencies between any teaching of any referencecited herein and the present specification, the latter shall prevail forpurposes of the invention.

The invention provides methods for enhancing the immune response causedby immunostimulatory compounds used for immunotherapy applications suchas, but not limited to, treatment of cancer, autoimmune disorders,asthma, respiratory allergies, food allergies, and bacteria, parasitic,and viral infections in adult and pediatric human and veterinaryapplications. Thus, the invention further provides compounds havingoptimal levels of immunostimulatory effect for immunotherapy and methodsfor making and using such compounds. In addition, compounds of theinvention are useful as adjuvants in combination with DNA vaccines,antibodies, and allergens; and in combination with chemotherapeuticagents and/or antisense oligonucleotides.

The present inventors have surprisingly discovered that modification ofan immunostimulatory oligonucleotide to optimally present its 5′ endsdramatically affects its immunostimulatory capabilities. In addition,the present inventors have discovered that the cytokine profile andspecies specificity of an immune response can be modulated by usingnovel purine or pyrimidine structures as part of an immunostimulatoryoligonucleotide.

In a first aspect, the invention provides immunostimulatoryoligonucleotides alone or comprising at least two oligonucleotideslinked at their 3′ ends, or an internucleoside linkage or afunctionalized nucleobase or sugar to a non-nucleotidic linker, at leastone of the oligonucleotides being an immunostimulatory oligonucleotideand having an accessible 5′ end. As used herein, the term “accessible 5′end” means that the 5′ end of the oligonucleotide is sufficientlyavailable such that the factors that recognize and bind tooligonucleotide and stimulate the immune system have access to it. Inoligonucleotides having an accessible 5′ end, the 5′ OH position of theterminal sugar is not covalently linked to more than two nucleosideresidues or any other moiety that interferes with interaction with the5′ end. Optionally, the 5′ OH can be linked to a phosphate,phosphorothioate, or phosphorodithioate moiety, an aromatic or aliphaticlinker, cholesterol, or another entity which does not interfere withaccessibility. The immunostimulatory oligonucleotides according to theinvention preferably further comprise an immunostimulatory dinucleotidecomprising a novel purine or pyrimidine.

In certain embodiments, the immunostimulatory oligonucleotides include aribozyme or a decoy oligonucleotide. As used herein, the term “ribozyme”refers to an oligonucleotide that possesses catalytic activity.Preferably, the ribozyme binds to a specific nucleic acid target andcleaves the target. As used herein, the term “decoy oligonucleotide”refers to an oligonucleotide that binds to a transcription factor in asequence-specific manner and arrests transcription activity. Preferably,the ribozyme or decoy oligonucleotide exhibits secondary structure,including, without limitation, stem-loop or hairpin structures. Incertain embodiments, at least one oligonucleotide comprisespoly(I)-poly(C). In certain embodiments, at least one set of Nn includesa string of 3 to 10 dGs and/or Gs or 2′-substituted ribo or arabino Gs.

For purposes of the invention, the term “oligonucleotide” refers to apolynucleoside formed from a plurality of linked nucleoside units. Sucholigonucleotides can be obtained from existing nucleic acid sources,including genomic or cDNA, but are preferably produced by syntheticmethods. In preferred embodiments each nucleoside unit includes aheterocyclic base and a pentofuranosyl, trehalose, arabinose,2′-deoxy-2′-substituted arabinose, 2′-O-substituted arabinose or hexosesugar group. The nucleoside residues can be coupled to each other by anyof the numerous known internucleoside linkages. Such internucleosidelinkages include, without limitation, phosphodiester, phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,acetamidate, carbamate, morpholino, borano, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone internucleoside linkages. The term“oligonucleotide” also encompasses polynucleosides having one or morestereospecific internucleoside linkage (e.g., (R_(P))- or(S_(P))-phosphorothioate, alkylphosphonate, or phosphotriesterlinkages). As used herein, the terms “oligonucleotide” and“dinucleotide” are expressly intended to include polynucleosides anddinucleosides having any such internucleoside linkage, whether or notthe linkage comprises a phosphate group. In certain preferredembodiments, these internucleoside linkages may be phosphodiester,phosphorothioate, or phosphorodithioate linkages, or combinationsthereof.

In some embodiments, the oligonucleotides each have from about 3 toabout 35 nucleoside residues, preferably from about 4 to about 30nucleoside residues, more preferably from about 4 to about 20 nucleosideresidues. In some embodiments, the immunostimulatory oligonucleotidescomprise oligonucleotides have from about 5 to about 18, or from about 5to about 14, nucleoside residues. As used herein, the term “about”implies that the exact number is not critical. Thus, the number ofnucleoside residues in the oligonucleotides is not critical, andoligonucleotides having one or two fewer nucleoside residues, or fromone to several additional nucleoside residues are contemplated asequivalents of each of the embodiments described above. In someembodiments, one or more of the oligonucleotides have 11 nucleotides. Inthe context of immunostimulatory oligonucleotides, preferred embodimentshave from about 13 to about 35 nucleotides, more preferably from about13 to about 26 nucleotides.

The term “oligonucleotide” also encompasses polynucleosides havingadditional substituents including, without limitation, protein groups,lipophilic groups, intercalating agents, diamines, folic acid,cholesterol and adamantane. The term “oligonucleotide” also encompassesany other nucleobase containing polymer, including, without limitation,peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups(PHONA), locked nucleic acids (LNA), morpholino-backboneoligonucleotides, and oligonucleotides having backbone sections withalkyl linkers or amino linkers.

The oligonucleotides of the invention can include naturally occurringnucleosides, modified nucleosides, or mixtures thereof. As used herein,the term “modified nucleoside” is a nucleoside that includes a modifiedheterocyclic base, a modified sugar moiety, or a combination thereof. Insome embodiments, the modified nucleoside is a non-natural pyrimidine orpurine nucleoside, as herein described. In some embodiments, themodified nucleoside is a 2′-substituted ribonucleoside anarabinonucleoside or a 2′-deoxy-2′-substituted-arabinoside.

For purposes of the invention, the term “2′-substituted ribonucleoside”or “2′-substituted arabinoside” includes ribonucleosides orarabinonucleoside in which the hydroxyl group at the 2′ position of thepentose moiety is substituted to produce a 2′-substituted or2′-O-substituted ribonucleoside. Preferably, such substitution is with alower alkyl group containing 1-6 saturated or unsaturated carbon atoms,or with an aryl group having 6-10 carbon atoms, wherein such alkyl, oraryl group may be unsubstituted or may be substituted, e.g., with halo,hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carboalkoxy, or amino groups. Examples of 2′-O-substitutedribonucleosides or 2′-β-substituted-arabinosides include, withoutlimitation 2′-O-methylribonucleosides or 2′-β-methylarabinosides and2′-O-methoxyethylribonucleosides or 2′-O-methoxyethylarabinosides.

The term “2′-substituted ribonucleoside” or “2′-substituted arabinoside”also includes ribonucleosides or arabinonucleosides in which the2′-hydroxyl group is replaced with a lower alkyl group containing 1-6saturated or unsaturated carbon atoms, or with an amino or halo group.Examples of such 2′-substituted ribonucleosides or 2′-substitutedarabinosides include, without limitation, 2′-amino, 2′-fluoro, 2′-allyl,and 2′-propargyl ribonucleosides or arabinosides.

The term “oligonucleotide” includes hybrid and chimericoligonucleotides. A “chimeric oligonucleotide” is an oligonucleotidehaving more than one type of internucleoside linkage. One preferredexample of such a chimeric oligonucleotide is a chimeric oligonucleotidecomprising a phosphorothioate, phosphodiester or phosphorodithioateregion and non-ionic linkages such as alkylphosphonate oralkylphosphonothioate linkages (see e.g., Pederson et al. U.S. Pat. Nos.5,635,377 and 5,366,878).

A “hybrid oligonucleotide” is an oligonucleotide having more than onetype of nucleoside. One preferred example of such a hybridoligonucleotide comprises a ribonucleotide or 2′-substitutedribonucleotide region, and a deoxyribonucleotide region (see, e.g.,Metelev and Agrawal, U.S. Pat. Nos. 5,652,355, 6,346,614 and 6,143,881).

For purposes of the invention, the term “immunostimulatoryoligonucleotide” refers to an oligonucleotide as described above thatinduces an immune response when administered to a vertebrate, such as afish, fowl, or mammal. As used herein, the term “mammal” includes,without limitation rats, mice, cats, dogs, horses, cattle, cows, pigs,rabbits, non-human primates, and humans. Useful immunostimulatoryoligonucleotides can be found described in Agrawal et al., WO 98/49288,published Nov. 5, 1998; WO 01/12804, published Feb. 22, 2001; WO01/55370, published Aug. 2, 2001; PCT/US01/13682, filed Apr. 30, 2001;and PCT/US01/30137, filed Sep. 26, 2001. Preferably, theimmunostimulatory oligonucleotide comprises at least one phosphodiester,phosphorothioate, or phosphorodithioate internucleoside linkage.

In some embodiments, the immunostimulatory oligonucleotide comprises animmunostimulatory dinucleotide of formula 5′-Pur*-Pur-3′, wherein Pur*is a natural or synthetic pyrimidine nucleoside and Pur is a natural orsynthetic purine nucleoside. In some preferred embodiments, theimmunostimulatory oligonucleotide comprises an immunostimulatorydinucleotide of formula 5′-Pur*-Pur-3′, wherein Pur* is a syntheticpurine nucleoside and Pur is a natural or synthetic purine nucleoside.In various places the dinucleotide is expressed as RpG, C*pG or YZ, inwhich case respectively, R, C*, or Y represents a synthetic purine. Aparticularly preferred synthetic purine is2-oxo-7-deaza-8-methyl-purine. When this synthetic purine is in the Pur*position of the dinucleotide, species-specificity (sequence dependence)of the immunostimulatory effect is overcome and cytokine profile isimproved. As used herein, the term “pyrimidine nucleoside” refers to anucleoside wherein the base component of the nucleoside is a monocyclicnucleobase. Similarly, the term “purine nucleoside” refers to anucleoside wherein the base component of the nucleoside is a bicyclicnucleobase. For purposes of the invention, a “synthetic” pyrimidine orpurine nucleoside includes a non-naturally occurring pyrimidine orpurine base, a non-naturally occurring sugar moiety, or a combinationthereof.

Preferred pyrimidine nucleosides according to the invention have thestructure (I):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, hydrogen bond acceptor, hydrophilic group, hydrophobic group,electron withdrawing group and electron donating group;

A is a hydrogen bond acceptor or a hydrophilic group;

A′ is selected from the group consisting of hydrogen bond acceptor,hydrophilic group, hydrophobic group, electron withdrawing group andelectron donating group;

X is carbon or nitrogen; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

Preferably, the sugar ring is derivatized with a phosphate moiety,modified phosphate moiety, or other linker moiety suitable for linkingthe pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH₂,—SH and —OH. Preferred hydrogen bond acceptors include, withoutlimitation, C═O, C═S, and the ring nitrogen atoms of an aromaticheterocycle, e.g., N3 of cytosine.

In some embodiments, the base moiety in (I) is a non-naturally occurringpyrimidine base. Examples of preferred non-naturally occurringpyrimidine bases include, without limitation, 5-hydroxycytosine,5-hydroxymethylcytosine, N4-alkylcytosine, preferably N4-ethylcytosine,and 4-thiouracil. However, in some embodiments 5-bromocytosine isspecifically excluded.

In some embodiments, the sugar moiety S′ in (I) is a non-naturallyoccurring sugar moiety. For purposes of the present invention, a“naturally occurring sugar moiety” is a sugar moiety that occursnaturally as part of nucleic acid, e.g., ribose and 2′-deoxyribose, anda “non-naturally occurring sugar moiety” is any sugar that does notoccur naturally as part of a nucleic acid, but which can be used in thebackbone for an oligonucleotide, e.g, hexose. Arabinose and arabinosederivatives are examples of preferred sugar moieties.

Preferred purine nucleoside analogs according to the invention have thestructure (II):

wherein:

D is a hydrogen bond donor;

D′ is selected from the group consisting of hydrogen, hydrogen bonddonor, and hydrophilic group;

A is a hydrogen bond acceptor or a hydrophilic group;

X is carbon or nitrogen;

each L is independently an atom selected from the group consisting of C,O, N and S; and

S′ is a pentose or hexose sugar ring, or a non-naturally occurringsugar.

Preferably, the sugar ring is derivatized with a phosphate moiety,modified phosphate moiety, or other linker moiety suitable for linkingthe pyrimidine nucleoside to another nucleoside or nucleoside analog.

Preferred hydrogen bond donors include, without limitation, —NH—, —NH₂,—SH and —OH. Preferred hydrogen bond acceptors include, withoutlimitation, C═O, C═S, —NO₂ and the ring nitrogen atoms of an aromaticheterocycle, e.g., N1 of guanine.

In some embodiments, the base moiety in (II) is a non-naturallyoccurring purine base. Examples of preferred non-naturally occurringpurine bases include, without limitation, 2-amino-6-thiopurine and2-amino-6-oxo-7-deazapurine. In some embodiments, the sugar moiety S′ in(II) is a naturally occurring sugar moiety, as described above forstructure (I).

In preferred embodiments, the immunostimulatory dinucleotide is selectedfrom the group consisting of CpG, C*pG, CpG*, and C*pG*, wherein thebase of C is cytosine, the base of C* is 2′-thymine, 5-hydroxycytosine,N4-alkyl-cytosine, 4-thiouracil or other non-natural pyrimidine, or2-oxo-7-deaza-8-methylpurine, wherein when the base is2-oxo-7-deaza-8-methyl-purine, it is preferably covalently bound to the1′-position of a pentose via the 1 position of the base; the base of Gis guanosine, the base of G* is 2-amino-6-oxo-7-deazapurine,2-oxo-7-deaza-8-methylpurine, 6-thioguanine, 6-oxopurine, or othernon-natural purine nucleoside, and p is an internucleoside linkageselected from the group consisting of phosphodiester, phosphorothioate,and phosphorodithioate. In certain preferred embodiments, theimmunostimulatory dinucleotide is not CpG.

The immunostimulatory oligonucleotides may include immunostimulatorymoieties on one or both sides of the immunostimulatory dinucleotide.Thus, in some embodiments, the immunostimulatory oligonucleotidecomprises an immunostimulatory domain of structure (III):

(III) 5′-Nn-N1-Y-Z-N1-Nn-3′wherein:

-   -   the base of Y is cytosine, thymine, 5-hydroxycytosine,        N4-alkyl-cytosine, 4-thiouracil or other non-natural pyrimidine        nucleoside, or 2-oxo-7-deaza-8 methyl purine, wherein when the        base is 2-oxo-7-deaza-8-methyl-purine, it is preferably        covalently bound to the 1′-position of a pentose via the 1        position of the base;    -   the base of Z is guanine, 2-amino-6-oxo-7-deazapurine,        2-oxo-7deaza-8-methylpurine, 2-amino-6-thio-purine, 6-oxopurine        or other non-natural purine nucleoside;

N1 and Nn, independent at each occurrence, is preferably a naturallyoccurring or a synthetic nucleoside or an immunostimulatory moietyselected from the group consisting of abasic nucleosides,arabinonucleosides, 2′-deoxyuridine, α-deoxyribonucleosides,β-L-deoxyribonucleosides, and nucleosides linked by a phosphodiester ormodified internucleoside linkage to the adjacent nucleoside on the 3′side, the modified internucleotide linkage being selected from, withoutlimitation, a linker having a length of from about 2 angstroms to about200 angstroms, C2-C18 alkyl linker, poly(ethylene glycol) linker,2-aminobutyl-1,3-propanediol linker, glyceryl linker, 2′-5′internucleoside linkage, and phosphorothioate, phosphorodithioate, ormethylphosphonate internucleoside linkage;

provided that at least one N1 or Nn is optionally an immunostimulatorymoiety;

wherein n is a number from 0 to 30; and

wherein the 3′ end, an internucleoside linker, or a derivatizednucleobase or sugar is linked directly or via a non-nucleotidic linkerto another oligonucleotide, which may or may not be immunostimulatory.

In some preferred embodiments, YZ is arabinocytidine or2′-deoxy-2′-substituted arabinocytidine and arabinoguanosine or 2′deoxy-2′-substituted arabinoguanosine. Preferred immunostimulatorymoieties include natural phosphodiester backbones and modifications inthe phosphate backbones, including, without limitation,methylphosphonates, methylphosphonothioates, phosphotriesters,phosphothiotriesters, phosphorothioates, phosphorodithioates, triesterprodrugs, sulfones, sulfonamides, sulfamates, formacetal,N-methylhydroxylamine, carbonate, carbamate, morpholino,boranophosphonate, phosphoramidates, especially primaryamino-phosphoramidates, N3 phosphoramidates and N5 phosphoramidates, andstereospecific linkages (e.g., (R_(P))- or (S_(P))-phosphorothioate,alkylphosphonate, or phosphotriester linkages).

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having sugar modifications, including, withoutlimitation, 2′-substituted pentose sugars including, without limitation,2′-O-methylribose, 2′-O-methoxyethylribose, 2′-O-propargylribose, and2′-deoxy-2′-fluororibose; 3′-substituted pentose sugars, including,without limitation, 3′-O-methylribose; 1′,2′-dideoxyribose; arabinose;substituted arabinose sugars, including, without limitation,1′-methylarabinose, 3′-hydroxymethylarabinose,4′-hydroxymethylarabinose, 3′-hydroxyarabinose and 2′-substitutedarabinose sugars; hexose sugars, including, without limitation,1,5-anhydrohexitol; and alpha-anomers. In embodiments in which themodified sugar is a 3′-deoxyribonucleoside or a 3′-O-substitutedribonucleoside, the immunostimulatory moiety is attached to the adjacentnucleoside by way of a 2′-5′ internucleoside linkage.

Preferred immunostimulatory moieties according to the invention furtherinclude oligonucleotides having other carbohydrate backbonemodifications and replacements, including peptide nucleic acids (PNA),peptide nucleic acids with phosphate groups (PHONA), locked nucleicacids (LNA), morpholino backbone oligonucleotides, and oligonucleotideshaving backbone linker sections having a length of from about 2angstroms to about 200 angstroms, including without limitation, alkyllinkers or amino linkers. The alkyl linker may be branched orunbranched, substituted or unsubstituted, and chirally pure or a racemicmixture. Most preferably, such alkyl linkers have from about 2 to about18 carbon atoms. In some preferred embodiments such alkyl linkers havefrom about 3 to about 9 carbon atoms. Some alkyl linkers include one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthioether. Some such functionalized alkyl linkers are poly(ethyleneglycol) linkers of formula —O—(CH₂—CH₂—O—)_(n) (n=1-9). Some otherfunctionalized alkyl linkers are peptides or amino acids.

Preferred immunostimulatory moieties according to the invention furtherinclude DNA isoforms, including, without limitation,β-L-deoxyribonucleosides and α-deoxyribonucleosides. Preferredimmunostimulatory moieties according to the invention incorporate 3′modifications, and further include nucleosides having unnaturalinternucleoside linkage positions, including, without limitation, 2′-5′,2′-2′,3′-3′ and 5′-5′ linkages.

Preferred immunostimulatory moieties according to the invention furtherinclude nucleosides having modified heterocyclic bases, including,without limitation, 5-hydroxycytosine, 5-hydroxymethylcytosine,N4-alkylcytosine, preferably N4-ethylcytosine, 4-thiouracil,6-thioguanine, 7-deazaguanine, inosine, nitropyrrole,C5-propynylpyrimidine, and diaminopurines, including, withoutlimitation, 2,6-diaminopurine.

By way of specific illustration and not by way of limitation, forexample, in the immunostimulatory domain of structure (III), amethylphosphonate internucleoside linkage at position N1 or Nn is animmunostimulatory moiety, a linker having a length of from about 2angstroms to about 200 angstroms, C2-C18 alkyl linker at position X1 isan immunostimulatory moiety, and a β-L-deoxyribonucleoside at positionX1 is an immunostimulatory moiety. See Table 1 below for representativepositions and structures of immunostimulatory moieties. It is to beunderstood that reference to a linker as the immunostimulatory moiety ata specified position means that the nucleoside residue at that positionis substituted at its 3′-hydroxyl with the indicated linker, therebycreating a modified internucleoside linkage between that nucleosideresidue and the adjacent nucleoside on the 3′ side. Similarly, referenceto a modified internucleoside linkage as the immunostimulatory moiety ata specified position means that the nucleoside residue at that positionis linked to the adjacent nucleoside on the 3′ side by way of therecited linkage.

TABLE 1 Position TYPICAL IMMUNOSTIMULATORY MOIETIES N1Naturally-occurring nucleosides, abasic nucleoside, arabinonucleoside,2′-deoxyuridine, β-L-deoxyribonucleoside C2-C18 alkyl linker,poly(ethylene glycol) linkage, 2-aminobutyl-1,3-propanediol linker(amino linker), 2′-5′ internucleoside linkage, methylphosphonateinternucleoside linkage Nn Naturally-occurring nucleosides, abasicnucleoside, arabinonucleosides, 2′-deoxyuridine, 2′-O-substitutedribonucleoside, 2′-5′ internucleoside linkage, methylphosphonateinternucleoside linkage, provided that N1 and N2 cannot both be abasiclinkages

Table 2 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving an upstream potentiation domain. As used herein, the term “Spacer9” refers to a poly(ethylene glycol) linker of formula—O—(CH₂CH₂—O)_(n)—, wherein n is 3. The term “Spacer 18” refers to apoly(ethylene glycol) linker of formula —O—(CH₂CH₂—O)_(n)—, wherein n is6. As used herein, the term “C2-C18 alkyl linker refers to a linker offormula —O—(CH₂)_(q)—O—, where q is an integer from 2 to 18.Accordingly, the terms “C3-linker” and “C3-alkyl linker” refer to alinker of formula —O—(CH₂)₃—O—. For each of Spacer 9, Spacer 18, andC2-C18 alkyl linker, the linker is connected to the adjacent nucleosidesby way of phosphodiester, phosphorothioate, or phosphorodithioatelinkages.

TABLE 2 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5′ N2Naturally-occurring nucleosides, 2-aminobutyl-1,3-propanediol linker 5′N1 Naturally-occurring nucleosides, β-L-deoxyribonucleoside, C2-C18alkyl linker, poly(ethylene glycol), abasic linker,2-aminobutyl-1,3-propanediol linker 3′ N1 Naturally-occurringnucleosides, 1′,2′-dideoxyribose, 2′-O-methyl- ribonucleoside, C2-C18alkyl linker, Spacer 9, Spacer 18 3′ N2 Naturally-occurring nucleosides,1′,2′-dideoxyribose, 3′- deoxyribonucleoside, β-L-deoxyribonucleoside,2′-O-propargyl- ribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer18, methylphosphonate internucleoside linkage 3′ N3 Naturally-occurringnucleosides, 1′,2′-dideoxyribose, C2-C18 alkyl linker, Spacer 9, Spacer18, methylphosphonate internucleoside linkage, 2′-5′ internucleosidelinkage, d(G)n, polyI-polyC 3′N 2 + 3′N 3 1′,2′-dideoxyribose,β-L-deoxyribonucleoside, C2-C18 alkyl linker, d(G)n, polyI-polyC 3′N3 +3′ N 4 2′-O-methoxyethyl-ribonucleoside, methylphosphonateinternucleoside linkage, d(G)n, polyI-polyC 3′N5 + 3′ N 61′,2′-dideoxyribose, C2-C18 alkyl linker, d(G)n, polyI-polyC 5′N1 + 3′ N3 1′,2′-dideoxyribose, d(G)n, polyI-polyC

Table 3 shows representative positions and structures ofimmunostimulatory moieties within an immunostimulatory oligonucleotidehaving a downstream potentiation domain.

TABLE 3 Position TYPICAL IMMUNOSTIMULATORY MOIETY 5′ N2methylphosphonate internucleoside linkage 5′ N1 methylphosphonateinternucleoside linkage 3′ N1 1′,2′-dideoxyribose, methylphosphonateinternucleoside linkage, 2′-O-methyl 3′ N2 1′,2′-dideoxyribose,β-L-deoxyribonucleoside, C2-C18 alkyl linker, Spacer 9, Spacer 18,2-aminobutyl-1,3-propanediol linker, methylphosphonate internucleosidelinkage, 2′-O-methyl 3′ N3 3′-deoxyribonucleoside, 3′-O-substitutedribonucleoside, 2′-O-propargyl-ribonucleoside 3′N2 + 3′ N31′,2′-dideoxyribose, β-L-deoxyribonucleoside

The immunostimulatory oligonucleotides according to the inventioncomprise at least two oligonucleotides linked at their 3′ ends orinternucleoside linkage or a functionalized nucleobase or sugar via anon-nucleotidic linker. For purposes of the invention, a“non-nucleotidic linker” is any moiety that can be linked to theoligonucleotides by way of covalent or non-covalent linkages. Preferablysuch linker is from about 2 angstroms to about 200 angstroms in length.Several examples of preferred linkers are set forth below. Non-covalentlinkages include, but are not limited to, electrostatic interaction,hydrophobic interactions, π-stacking interactions, and hydrogen bonding.The term “non-nucleotidic linker” is not meant to refer to aninternucleoside linkage, as described above, e.g., a phosphodiester,phosphorothioate, or phosphorodithioate functional group, that directlyconnects the 3′-hydroxyl groups of two nucleosides. For purposes of thisinvention, such a direct 3′-3′ linkage (no linker involved) isconsidered to be a “nucleotidic linkage.”

In some embodiments, the non-nucleotidic linker is a metal, including,without limitation, gold particles. In some other embodiments, thenon-nucleotidic linker is a soluble or insoluble biodegradable polymerbead.

In yet other embodiments, the non-nucleotidic linker is an organicmoiety having functional groups that permit attachment to theoligonucleotide. Such attachment preferably is by any stable covalentlinkage. As a non-limiting example, the linker may be attached to anysuitable position on the nucleoside, as illustrated in FIG. 5. In somepreferred embodiments, the linker is attached to the 3′-hydroxyl. Insuch embodiments, the linker preferably comprises a hydroxyl functionalgroup, which preferably is attached to the 3′-hydroxyl by means of aphosphodiester, phosphorothioate, phosphorodithioate ornon-phosphate-based linkages.

In some embodiments, the non-nucleotidic linker is a biomolecule,including, without limitation, polypeptides, antibodies, lipids,antigens, allergens, and oligosaccharides. In some other embodiments,the non-nucleotidic linker is a small molecule. For purposes of theinvention, a small molecule is an organic moiety having a molecularweight of less than 1,000 Da. In some embodiments, the small moleculehas a molecular weight of less than 750 Da.

In some embodiments, the small molecule is an aliphatic or aromatichydrocarbon, either of which optionally can include, either in thelinear chain connecting the oligonucleotides or appended to it, one ormore functional groups selected from the group consisting of hydroxy,amino, thiol, thioether, ether, amide, thioamide, ester, urea, andthiourea. The small molecule can be cyclic or acyclic. Examples of smallmolecule linkers include, but are not limited to, amino acids,carbohydrates, cyclodextrins, adamantane, cholesterol, haptens andantibiotics. However, for purposes of describing the non-nucleotidiclinker, the term “small molecule” is not intended to include anucleoside.

In some embodiments, the small molecule linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4, or from1 to about 3. In some other embodiments, the small molecule linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6, or from 2 to about 4.

Some non-nucleotidic linkers according to the invention permitattachment of more than two oligonucleotides. For example, the smallmolecule linker glycerol has three hydroxyl groups to whicholigonucleotides may be covalently attached. Some immunostimulatoryoligonucleotides according to the invention, therefore, comprise morethan two oligonucleotides linked at their 3′ ends to a non-nucleotidiclinker.

The immunostimulatory oligonucleotides of the invention may convenientlybe synthesized using an automated synthesizer and phosphoramiditeapproach as schematically depicted in FIGS. 3 and 4, and furtherdescribed in the Examples. In some embodiments, the immunostimulatoryoligonucleotides are synthesized by a linear synthesis approach (seeFIG. 3). As used herein, the term “linear synthesis” refers to asynthesis that starts at one end of the immunostimulatoryoligonucleotide and progresses linearly to the other end. Linearsynthesis permits incorporation of either identical or un-identical (interms of length, base composition and/or chemical modificationsincorporated) monomeric units into the immunostimulatoryoligonucleotides.

An alternative mode of synthesis is “parallel synthesis”, in whichsynthesis proceeds outward from a central linker moiety (see FIG. 4). Asolid support attached linker can be used for parallel synthesis, as isdescribed in U.S. Pat. No. 5,912,332. Alternatively, a universal solidsupport (such as phosphate attached controlled pore glass) support canbe used.

Parallel synthesis of immunostimulatory oligonucleotides has severaladvantages over linear synthesis: (1) parallel synthesis permits theincorporation of identical monomeric units; (2) unlike in linearsynthesis, both (or all) the monomeric units are synthesized at the sametime, thereby the number of synthetic steps and the time required forthe synthesis is the same as that of a monomeric unit; and (3) thereduction in synthetic steps improves purity and yield of the finalimmunostimulatory oligonucleotide product.

At the end of the synthesis by either linear synthesis or parallelsynthesis protocols, the immunostimulatory oligonucleotides mayconveniently be deprotected with concentrated ammonia solution or asrecommended by the phosphoramidite supplier, if a modified nucleoside isincorporated. The product immunostimulatory oligonucleotide ispreferably purified by reversed phase HPLC, detritylated, desalted anddialyzed.

Table 4 shows representative immunostimulatory oligonucleotidesaccording to the invention.

TABLE 4 Examples of Immunostimulatory Oligonucleotides Sequences SEQ IDNO. Sequences and Modification  1 5′-TCTGTR′GTTCT-X-TCTTGR′TGTCT-5′  25′-ACACACCAACT-X-TCAACCACACA-5′ (Control)  35′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′  45′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′  55′-CTGTR GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′  65′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′  75′-TCTGTR′GTTCT-X-CGTTCGAACGT-5 ′  8 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ 9 5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′ 105′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ 11 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′12 5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ 135′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ 145′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ 155′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ 16 5′-TCRAACRTTCR-X-RCTTRCAARCT-5′R′ = 1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁ = 2′-O-methyl-ribonucleotides; R =2′-deoxy-7-deazaguanosine; X = glycerol linker.

In a second aspect, the invention provides immunostimulatoryoligonucleotide conjugates comprising an immunostimulatoryoligonucleotide, as described above, and an antigen conjugated to theimmunostimulatory oligonucleotide at a position other than theaccessible 5′ end. In some embodiments, the non-nucleotidic linkercomprises an antigen, which is conjugated to the oligonucleotide. Insome other embodiments, the antigen is conjugated to the oligonucleotideat a position other than its 3′ end. In some embodiments, the antigenproduces a vaccine effect.

The antigen is preferably selected from the group consisting of antigensassociated with a pathogen, antigens associated with a cancer, antigensassociated with an auto-immune disorder, and antigens associated withother diseases such as, but not limited to, veterinary or pediatricdiseases. For purposes of the invention, the term “associated with”means that the antigen is present when the pathogen, cancer, auto-immunedisorder, food allergy, respiratory allergy, asthma or other disease ispresent, but either is not present, or is present in reduced amounts,when the pathogen, cancer, auto-immune disorder, food allergy,respiratory allergy, or disease is absent.

The immunostimulatory oligonucleotide is covalently linked to theantigen, or it is otherwise operatively associated with the antigen. Asused herein, the term “operatively associated with” refers to anyassociation that maintains the activity of both immunostimulatoryoligonucleotide and antigen. Nonlimiting examples of such operativeassociations include being part of the same liposome or other suchdelivery vehicle or reagent. In embodiments wherein theimmunostimulatory oligonucleotide is covalently linked to the antigen,such covalent linkage preferably is at any position on theimmunostimulatory oligonucleotide other than an accessible 5′ end of animmunostimulatory oligonucleotide. For example, the antigen may beattached at an internucleoside linkage or may be attached to thenon-nucleotidic linker. Alternatively, the antigen may itself be thenon-nucleotidic linker.

In a third aspect, the invention provides pharmaceutical formulationscomprising an immunostimulatory oligonucleotide or immunostimulatoryoligonucleotide conjugate according to the invention and aphysiologically acceptable carrier. As used herein, the term“physiologically acceptable” refers to a material that does notinterfere with the effectiveness of the immunostimulatoryoligonucleotide and is compatible with a biological system such as acell, cell culture, tissue, or organism. Preferably, the biologicalsystem is a living organism, such as a vertebrate.

As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, or other materialwell known in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication.

The preparation of pharmaceutically acceptable formulations containingthese materials is described in, e.g., Remington's PharmaceuticalSciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton,Pa., 1990.

In a fourth aspect, the invention provides methods for generating animmune response in a vertebrate, such methods comprising administeringto the vertebrate an immunostimulatory oligonucleotide orimmunostimulatory oligonucleotide conjugate according to the invention.In some embodiments, the vertebrate is a mammal. For purposes of thisinvention, the term “mammal” is expressly intended to include humans. Inpreferred embodiments, the immunostimulatory oligonucleotide orimmunostimulatory oligonucleotide conjugate is administered to avertebrate in need of immunostimulation.

In the methods according to this aspect of the invention, administrationof immunostimulatory oligonucleotide or immunostimulatoryoligonucleotide conjugate can be by any suitable route, including,without limitation, parenteral, oral, sublingual, transdermal, topical,intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal,by gene gun, dermal patch or in eye drop or mouthwash form.Administration of the therapeutic compositions of immunostimulatoryoligonucleotides can be carried out using known procedures at dosagesand for periods of time effective to reduce symptoms or surrogatemarkers of the disease. When administered systemically, the therapeuticcomposition is preferably administered at a sufficient dosage to attaina blood level of immunostimulatory oligonucleotide from about 0.0001micromolar to about 10 micromolar. For localized administration, muchlower concentrations than this may be effective, and much higherconcentrations may be tolerated. Preferably, a total dosage ofimmunostimulatory oligonucleotide ranges from about 0.001 mg per patientper day to about 200 mg per kg body weight per day. It may be desirableto administer simultaneously, or sequentially a therapeuticallyeffective amount of one or more of the therapeutic compositions of theinvention to an individual as a single treatment episode.

In certain preferred embodiments, immunostimulatory oligonucleotide orimmunostimulatory oligonucleotide conjugate according to the inventionare administered in combination with vaccines, antibodies, cytotoxicagents, allergens, antibiotics, antisense oligonucleotides, peptides,proteins, gene therapy vectors, DNA vaccines and/or adjuvants to enhancethe specificity or magnitude of the immune response. In theseembodiments, the immunostimulatory oligonucleotides of the invention canvariously act as adjuvants and/or produce direct immunostimulatoryeffects.

Either the immunostimulatory oligonucleotide or immunostimulatoryoligonucleotide conjugate or the vaccine, or both, may optionally belinked to an immunogenic protein, such as keyhole limpet hemocyanin(KLH), cholera toxin B subunit, or any other immunogenic carrierprotein. Any of the plethora of adjuvants may be used including, withoutlimitation, Freund's complete adjuvant, KLH, monophosphoryl lipid A(MPL), alum, and saponins, including QS-21, imiquimod, R848, orcombinations thereof.

For purposes of this aspect of the invention, the term “in combinationwith” means in the course of treating the same disease in the samepatient, and includes administering the immunostimulatoryoligonucleotide and/or the vaccine and/or the adjuvant in any order,including simultaneous administration, as well as temporally spacedorder of up to several days apart. Such combination treatment may alsoinclude more than a single administration of the immunostimulatoryoligonucleotide, and/or independently the vaccine, and/or independentlythe adjuvant. The administration of the immunostimulatoryoligonucleotide and/or vaccine and/or adjuvant may be by the same ordifferent routes.

The methods according to this aspect of the invention are useful formodel studies of the immune system. The methods are also useful for theprophylactic or therapeutic treatment of human or animal disease. Forexample, the methods are useful for pediatric and veterinary vaccineapplications.

In a fifth aspect, the invention provides methods for therapeuticallytreating a patient having a disease or disorder, such methods comprisingadministering to the patient an immunostimulatory oligonucleotide orimmunostimulatory oligonucleotide conjugate according to the invention.In various embodiments, the disease or disorder to be treated is cancer,an autoimmune disorder, airway inflammation, inflammatory disorders,allergy, asthma or a disease caused by a pathogen. Pathogens includebacteria, parasites, fungi, viruses, viroids and prions. Administrationis carried out as described for the fourth aspect of the invention.

For purposes of the invention, the term “allergy” includes, withoutlimitation, food allergies and respiratory allergies. The term “airwayinflammation” includes, without limitation, asthma. As used herein, theterm “autoimmune disorder” refers to disorders in which “self” proteinsundergo attack by the immune system. Such term includes autoimmuneasthma.

In any of the methods according to this aspect of the invention, theimmunostimulatory oligonucleotide or immunostimulatory oligonucleotideconjugate can be administered in combination with any other agent usefulfor treating the disease or condition that does not diminish theimmunostimulatory effect of the immunostimulatory oligonucleotide. Forexample, in the treatment of cancer, it is contemplated that theimmunostimulatory oligonucleotide or immunostimulatory oligonucleotideconjugate may be administered in combination with a chemotherapeuticcompound.

The examples below are intended to further illustrate certain preferredembodiments of the invention, and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1 Synthesis of Oligonucleotides ContainingImmunostimulatory Moieties

Oligonucleotides were synthesized on a 1 μmol to 0.1 mM scale using anautomated DNA synthesizer (OligoPilot II, AKTA, (Amersham) and/orExpedite 8909 (Applied Biosystem)), following the linear synthesis orparallel synthesis procedures outlined in FIGS. 3 and 4.

5′-DMT dA, dG, dC and T phosphoramidites were purchased from Proligo(Boulder, Colo.). 5′-DMT 7-deaza-dG and araG phosphoramidites wereobtained from Chemgenes (Wilmington, Mass.). DiDMT-glycerol linker solidsupport was obtained from Chemgenes.1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine amidite wasobtained from Glen Research (Sterling, Va.), 2′-O-methylribonuncleosideamidites were obtained from Promega (Obispo, Calif.). Alloligonucleotides were phosphorothioate backbone modified.

All nucleoside phosphoramidites were characterized by ³¹P and ¹H NMRspectra. Modified nucleosides were incorporated at specific sites usingnormal coupling cycles recommended by the supplier. After synthesis,oligonucleotides were deprotected using concentrated ammonium hydroxideand purified by reverse phase HPLC, detritylation, followed by dialysis.Purified oligonucleotides as sodium salt form were lyophilized prior touse. Purity was tested by CGE and MALDI-TOF MS. Endotoxin levels weredetermined by LAL test and were below 1.0 EU/mg.

Example 2 Activity of Short-Immunostimulatory Oligonucleotides in MurineSpleen Cell Cultures

C57/BL6 spleen cells were cultured with indicated concentrations ofcompounds. After 24 hours the supernatants were collected and the levelsof IL-12 and IL-6 were determined by ELISA. All immunostimulatoryoligonucleotides showed a concentration-dependent induction of twotypical cytokines, IL-12 and IL-6 (FIGS. 6-7).

Example 3 Need Protocol for IFN-Alpha Induction in Human pDC

Peripheral blood mononuclear cells (PBMCs) from freshly drawn healthyvolunteer blood (CBR Laboratories, Boston, Mass.) were isolated byFicoll density gradient centrifugation method (Histopaque-1077, Sigma).pDCs were isolated from PBMCs by positive selection using the BDCA4 cellisolation kits (Miltenyi Biotec) according to the manufacturer'sinstructions. pDCs were plated in 96-well dishes using 1×10⁶ cells/ml.The IMOs dissolved in DPBS (pH 7.4; Mediatech) were added to a finalconcentration of 10.0 μg/ml to the cell cultures. The cells were thenincubated at 37° C. for 24 hr and the supernatants were collected forELISA assays. The experiments were performed in triplicate wells. Thelevels of IFN-α were measured by sandwich ELISA. The required reagents,including cytokine antibodies and standards, were purchased fromPharMingen.

Example 4 IFN-Alpha Induction in Human PMBC

Human PBMCs were plated in 48-well plates using 5×10⁶ cells/ml. The IMOsdissolved in DPBS (pH 7.4; Mediatech) were added to a finalconcentration of 10.0 μg/ml to the cell cultures. The cells were thenincubated at 37° C. for 24 hr and the supernatants were collected forELISA assays. The experiments were performed in triplicate wells. Thelevels of IFN-α were measured by sandwich ELISA. The required reagents,including cytokine antibodies and standards, were purchased fromPharMingen.

Example 5 Human B-Cell Proliferation

The culture medium used for the assay consisted of RPMI 1640 mediumsupplemented with 1.5 mM glutamine, 1 mM sodium pyruvate, 0.1 mMnon-essential amino acids, 50 μM 2-mercaptoethanol, 100 IU/mlpenicillin-streptomycin mix and 10% heat-inactivated fetal bovine serum.A total of 0.5×10⁶ B cells per ml (i.e. 1×10⁵/200 μl/well) werestimulated in 96 well flat bottom plates with different concentrationsof test oligonucleotides in triplicate for a total period of 72 hours.After 66 h, cells were pulsed with 0.75 μCi of [³H]-thymidine (1Ci=37GBq; Perkin Elmer Life Sciences) in 20 μl RPMI 1640 medium (no serum)per well and harvested 8 h later. The plates were then harvested using acell harvester and radioactive incorporation was determined usingstandard liquid scintillation technique. The results are expressedeither as mean cpm+/−SD or as proliferation index (cpm treated group/cpmmedium control).

EQUIVALENTS

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1-30. (canceled)
 31. A method for therapeutically treating a vertebratehaving cancer, an autoimmune disorder, airway inflammation, inflammatorydisorders, skin disorders, allergy, asthma or a disease caused by apathogen, such method comprising administering to the patient animmunostimulatory oligonucleotide having a structure from the group of5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ IDNO: 3-5′); 5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO:4-3′-X-3′-SEQ ID NO: 4-5′); 5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′(5′-SEQ ID NO: 5-3′-X-3′-SEQ ID NO: 5-5′);5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ IDNO: 6-5′); 5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQID NO 17-5′); 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO:8-3′-X-3′-SEQ ID NO: 8-5′); 5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′(5′-SEQ ID NO: 9-3′-X-3′-SEQ ID NO: 9-5′);5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO:10-5′); 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQID NO: 11-5′); 5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO:12-3′-X-3′-SEQ ID NO: 12-5′); 5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′(5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO: 13-5′);5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO:14-5′); and 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO:15-3′-X-3′-SEQ ID NO: 15-5′), whereinR′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁=2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine andX=glycerol linker.
 32. The method according to claim 31, wherein theroute of administration is selected from parenteral, oral, sublingual,transdermal, topical, intranasal, aerosol, intraocular, intratracheal,intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.33. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ IDNO: 3-5′).
 34. The method according to claim 31 comprising administeringan immunostimulatory oligonucleotide having the structure5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO: 4-3′-X-3′-SEQ ID NO:4-5′).
 35. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′ (5′-SEQ ID NO: 5-3′-X-3′-SEQ IDNO: 5-5′).
 36. The method according to claim 31 comprising administeringan immunostimulatory oligonucleotide having the structure5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ IDNO: 6-5′).
 37. The method according to claim 31 comprising administeringan immunostimulatory oligonucleotide having the structure5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQ ID NO:17-5′).
 38. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO: 8-3′-X-3′-SEQ ID NO:8-5′).
 39. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′ (5′-SEQ ID NO: 9-3′-X-3′-SEQ IDNO: 9-5′).
 40. The method according to claim 31 comprising administeringan immunostimulatory oligonucleotide having the structure5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO:10-5′).
 41. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQ ID NO:11-5′).
 42. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO: 12-3′-X-3′-SEQ IDNO: 12-5′).
 43. The method according to claim 31 comprisingadministering an immunostimulatory oligonucleotide having the structure5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ (5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO:13-5′).
 44. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO:14-5′).
 45. The method according to claim 31 comprising administering animmunostimulatory oligonucleotide having the structure5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO: 15-3′-X-3′-SEQ ID NO:15-5′).
 46. A method for preventing cancer, an autoimmune disorder,airway inflammation, inflammatory disorders, skin disorders, allergy,asthma or a disease caused by a pathogen in a vertebrate, such methodcomprising administering to the vertebrate an immunostimulatoryoligonucleotide having a structure from the group of5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ IDNO: 3-5′); 5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO:4-3′-X-3′-SEQ ID NO: 4-5′); 5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′(5′-SEQ ID NO: 5-3′-X-3′-SEQ ID NO: 5-5′);5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ IDNO: 6-5′); 5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQID NO: 17-5′); 5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO:8-3′-X-3′-SEQ ID NO: 8-5′); 5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′(5′-SEQ ID NO: 9-3′-X-3′-SEQ ID NO: 9-5′);5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO:10-5′); 5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQID NO: 11-5′); 5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO:12-3′-X-3′-SEQ ID NO: 12-5′); 5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′(5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO: 13-5′);5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO:14-5′); and 5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO:15-3′-X-3′-SEQ ID NO: 15-5′), whereinR′=1-(2′-deoxy-β-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine;A₁/C₁/G₁/U₁=2′-O-methyl-ribonucleotides; R=2′-deoxy-7-deazaguanosine andX=glycerol linker.
 47. The method according to claim 46, wherein theroute of administration is selected from parenteral, oral, sublingual,transdermal, topical, intranasal, aerosol, intraocular, intratracheal,intrarectal, vaginal, gene gun, dermal patch, eye drop and mouthwash.48. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCTGTR′GTTC₁U₁-X-U₁C₁TTGR′TGTCT-5′ (5′-SEQ ID NO: 3-3′-X-3′-SEQ IDNO: 3-5′).
 49. The method according to claim 46 comprising administeringan immunostimulatory oligonucleotide having the structure5′-CTGTR′GTTCTC-X-CTCTTGR′TGTC-5′ (5′-SEQ ID NO: 4-3′-X-3′-SEQ ID NO:4-5′).
 50. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-CTGTR′GTTCU₁C₁-X-C₁U₁CTTGR′TGTC-5′ (5′-SEQ ID NO: 5-3′-X-3′-SEQ IDNO: 5-5′).
 51. The method according to claim 46 comprising administeringan immunostimulatory oligonucleotide having the structure5′-CTGTR′GTTC₁U₁C₁-X-C₁U₁C₁TTGR′TGTC-5′ (5′-SEQ ID NO: 6-3′-X-3′-SEQ IDNO: 6-5′).
 52. The method according to claim 46 comprising administeringan immunostimulatory oligonucleotide having the structure5′-TCTGTR′GTTCT-X-CGTTCGAACGT-5′ (5′-SEQ ID NO: 7-3′-X-3′-SEQ ID NO:17-5′).
 53. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCTGTR′GACAG-X-GACAGR′TGTCT-5′ (5′-SEQ ID NO: 8-3′-X-3′-SEQ ID NO:8-5′).
 54. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCTGTR′GACA₁G₁-X-G₁A₁CAGR′TGTCT-5′ (5′-SEQ ID NO: 9-3′-X-3′-SEQ IDNO: 9-5′).
 55. The method according to claim 46 comprising administeringan immunostimulatory oligonucleotide having the structure5′-TCAGTR′GTTAG-X-GATTGR′TGACT-5′ (5′-SEQ ID NO: 10-3′-X-3′-SEQ ID NO:10-5′).
 56. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TCAGTR′GACTG-X-GTCAGR′TGACT-5′ (5′-SEQ ID NO: 11-3′-X-3′-SEQ ID NO:11-5′).
 57. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TR′GTR′GAR′GAT-X-TAGR′AGR′TGR′T-5′ (5′-SEQ ID NO: 12-3′-X-3′-SEQ IDNO: 12-5′).
 58. The method according to claim 46 comprisingadministering an immunostimulatory oligonucleotide having the structure5′-TR′GTR′GTAGTA-X-ATGATGR′TGR′T-5′ (5′-SEQ ID NO: 13-3′-X-3′-SEQ ID NO:13-5′).
 59. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TR′GAAR′GTTCT-X-TCTTGR′AAGR′T-5′ (5′-SEQ ID NO: 14-3′-X-3′-SEQ ID NO:14-5′).
 60. The method according to claim 46 comprising administering animmunostimulatory oligonucleotide having the structure5′-TR′GTAR′GTACT-X-TCATGR′ATGR′T-5′ (5′-SEQ ID NO: 15-3′-X-3′-SEQ ID NO:15-5′). 61-63. (canceled)
 64. The method according to claim 31, furthercomprising administering an antibody, antisense oligonucleotide,protein, antigen, allergen, chemotherapeutic agent or adjuvant.
 65. Themethod according to claim 46, further comprising administering anantibody, antisense oligonucleotide, protein, antigen, allergen,chemotherapeutic agent or adjuvant.